Prostate-specific antigen (PSA) is a kallikrein like serine protease secreted by the epithelial cells of the prostate gland. It is one of the predominant proteins present in prostatic fluid. Low levels of PSA are detected in all males, although the physiological function of PSA in the blood circulation still is not clear. Changes in anatomy of the prostate gland may lead to the diffusion of PSA into the blood circulation. Elevated PSA concentration in serum is indicative of several pathologic conditions including benign prostatic hyperplasia (BPH), prostatitis and prostate cancer.
If an active protease remains uncontrolled in circulation, it may potentially damage cellular components. The proteolytic activity of PSA in serum is inhibited by protease inhibitors such as alpha-1-antichymotrypsin (xcex11-ACT), alpha-2-macroglobulin (xcex12-M), and other acute phase proteins. The predominant form of PSA in prostate cancer patient serum (xcx9c86% of total), as detected by the current immunoassays, is complexed with xcex11-ACT. The rest of the immunologically detectable PSA exists as an enzymatically non-reactive free form. In most prostate cancer patients, the PSA level is consistently higher than that of normal males. As PSA is produced primarily by the prostate gland, its serum value in prostate cancer patients is reduced to virtually non-detectable levels after successful radical prostatectomy. Therefore, PSA is currently recognized as an important in vitro diagnostic parameter for monitoring patients with prostate cancer in order to detect residual disease and recurrence after therapeutic and/or surgical intervention.
xcex12-M possesses the ability to inhibit proteineases displaying different specificities and catalytic mechanisms. Cleavage of a peptide bond within the bait region triggers a conformational change that encages the proteinase and sterically hinders its access to larger substrates and active-site-directed inhibitors, as well as to antibodies. It has been demonstrated that xcex12-M is the major inhibitor of proteolytic activity of PSA. Honda et al., Clin. Chem. vol. 42, pp 1785-1788 (1996). The current generation of total PSA assays do not recognize PSA-xcex12-M complex, and technical difficulties in measuring this complex in patient""s serum have undermined the clinical significance of PSA-xcex12-M complex. Currently the proportion of free to total PSA is being used to discriminate BPH and prostate cancer. A higher proportion of free PSA is indicative of a high probability of BPH.
Prostate cancer is the second leading cause of death in the U.S. male population. Currently PSA is used for the early diagnosis and monitoring of prostate cancer. There is significant overlap between the PSA serum level values obtained from patients afflicted with benign conditions, such as benign prostatic hyperplasia (BPH), and those afflicted with prostate cancer. Therefore, PSA alone cannot be used as a reliable parameter to a screen for prostate cancer, or to discriminate BPH from prostate cancer.
It is an object of the invention to provide methods and diagnostic compositions capable of accurately determining the levels of PSA-xcex12-macroglobulin complex in patient blood samples as a reliable indicator of the presence and/or the stage of prostate cancer.
The present invention is based on the discovery by Applicants that PSA-xcex12-M complexes exist in clinically significant concentrations in fluid samples drawn from individuals afflicted with prostate cancer. Applicants for the first time have shown that these complexes are present in vivo, and have detected the presence and concentration of the complexes in the sera of prostate cancer patients using molecular sieve chromatography, Western blot analysis and a two-site immunoassay. Applicants have demonstrated that as much as 60% of the total circulating levels of PSA exist as the PSA-xcex12-M complex in prostate cancer patients. Measurement of the levels of PSA-xcex12-M complexes in biological fluids provide a better indicator of the presence of prostate cancer, as well as an earlier indication of malignancy, than measuring the PSA-ACT complex or free PSA. Measurement of the PSA-xcex12-M complex can detect false negatives which occur using PSA tests, and improve clinical sensitivity. Measurement of PSA-xcex12-M complexes also can be used in screening for and diagnosing prostate cancer, to differentiate between benign conditions such as BPH and prostate cancer, for the early detection of prostate cancer, as an indicator of metastases and for differentiating between latent and potentially aggressive prostate cancer.
In one aspect, the invention comprises methods for detecting the presence and/or determining the quantity of PSA-xcex12-M complex in a biological fluid of a subject. The method generally comprises contacting the sample of biological fluid with one or more binding proteins that specifically bind to the PSA-xcex12-M complex, and detecting the binding proteins bound to said complex. The subject preferably is a human, and the biological fluid preferably is blood serum or plasma, or a fraction thereof. In a preferred embodiment, the method is a two-site immunoassay. The immunoassay protocol comprises combining serum from a subject suspected of having or at risk for prostate cancer with a binding protein specific for PSA under conditions sufficient to promote binding of the binding protein to PSA in the sample. The mixture then is combined with a second binding protein which is specific for xcex12-M in the sample. The amount of PSA-xcex12-M in the serum sample then can be determined by detecting any material to which has bound both binding proteins.
The invention further comprises methods for detecting the presence of or determining the quantity of PSA-xcex12-M complex in a biological fluid of a subject using a Western blot method. In this embodiment, the biological fluid, or fraction thereof, is solubilized with a detergent, preferably sodium dodecyl sulfate (SDS), then applied to gels. Electrophoresis is performed to separate the components. The separated components then are transferred to a solid substrate, such as a nitrocellulose membrane. In a preferred embodiment, a blocking agent is applied to the substrate to block non-specific binding. The components then are immunodetected with monoclonal antibodies against PSA. The amount of the complex present then can be quantitated, for example, using quantitative densitometry.
In another aspect, the invention comprises methods for detecting the presence of prostate cancer in a subject by detecting the presence of PSA-xcex12-M complex in a biological fluid of a subject. In preferred embodiments of these methods, the PSA-xcex12-M complex is detected using either the Western blot method or the immunoassay method described above.
The invention further comprises methods for differentiating between benign prostate conditions and prostate cancer, or to determine the stage of prostate cancer. This method comprises quantitating the amount of PSA-xcex12-M complex in the biological fluid of a subject, and correlating the level of the complex with the stage of the condition or cancer. In preferred embodiments, the amount of complex in the biological fluid is determined by Western blot or immunoassay methods. In a particularly preferred embodiment, the serum levels of PSA-xcex12-M complex are determined by obtaining a serum sample from an individual afflicted with or at risk for prostate cancer, combining the serum sample with a first conjugate comprising a PSA-specific binding protein and a solid phase under conditions sufficient to induce reaction between the first conjugate and PSA in the sample. The mixture formed after this step then is contacted with a second conjugate comprising a binding protein specific for xcex12-M in the sample and a detectable moiety under conditions sufficient to induce reaction between the second conjugate and xcex12-M in the sample. The solid phase, which at this point is attached to a reaction product comprising PSA specific binding protein, PSA-xcex12-M complex and xcex12-M-specific binding protein with a detectable moiety, is separated from the reaction mixture. The amount of PSA-xcex12-M complex then can be determined based on the amount of detectable moiety.
Applicants have found that substantial concentrations of PSA-xcex12-M complex are present in the sera of prostate cancer patients. The amount of PSA-xcex12-M complex is indicative of the stage or aggressiveness of the cancer. Thus, in addition to detecting the presence of prostate cancer, another aspect of the invention comprises methods for determining the stage of any cancer which is detected, for monitoring the progress of prostate cancer in a patient, and for differentiating between prostate cancer and benign states, such as BPH.
The invention further comprises kits for performing the methods. In one embodiment, the kit comprises, at a minimum, a first conjugate comprising a PSA-specific binding protein and a separable tag or solid phase; and a second conjugate comprising a binding protein which is specific for xcex12-M and a detectable moiety. The kit optionally may contain various reagents and containers, or other components useful for carrying out the method.
The present invention provides improved methods and kits for detecting the presence of, monitoring, and determining the stage of prostate cancer in an individual based on the discovery that the presence and/or amount of PSA-xcex12-M complex in the biological fluids of a subject is indicative of the presence and stage of prostate cancer. The methods and kits of the invention also provide a means to distinguish between prostate cancer and a benign prostate condition, such as BPH.